Project Summary Disrupted energy metabolism is a central driver of dysfunction and death in a wide range of diseases, and may also contribute to aging. Energy failure, or insufficient energy to support normal function, can lead to neurodegenerative diseases, ischemia and heart failure, while an imbalance in energy production may contribute to cancer. As such, boosting energy levels has great therapeutic potential to improve cellular function and survival. However, there are only anecdotal examples of how to increase or preserve cellular ATP levels. To address this critical unmet need for anti-aging and energy-focused therapeutics, we developed an innovative high throughput screen that uses a fluorescent biosensor to measure ATP levels in individual cells, and used it to identify genes and pathways that regulate ATP levels. In this proposal, we will test our central hypothesis that increasing ATP can enhance the function and survival of vulnerable cells, but the efficacy depends on the mechanism by which ATP is increased. The overall objectives of our proposed study are to define the most robust mechanisms to increase cellular energy levels, and to determine if increasing ATP boosts the function and survival of human cells that are susceptible to diseases of energy failure. We will accomplish these objectives in two specific aims. (1) We will use metabolomics, transcriptomics and assays of energy production and consumption to determine the broad mechanisms by which energy levels can be increased. (2) We will determine if increasing the ATP level can enhance the function and survival of human neurons and cardiomyocytes. Overall, these studies will i) determine the broad cellular mechanisms by which cellular energy levels can be increased, and ii) begin to assess the therapeutic potential of increasing energy levels to protect against energy failure in metabolically vulnerable human cells.